Highlights d A CRISPR interference platform for genetic screens in human iPSC-derived neurons d Survival screens uncover genes essential for neurons, but not iPSCs or cancer cells d Single-cell RNA-seq screens reveal distinct neuronal roles for ubiquitous genes d Arrayed high-content screens uncover genes controlling neuronal morphology
Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.
SummaryLowering total tau levels is an attractive therapeutic strategy for Alzheimer's disease and other tauopathies. High-throughput screening in neurons derived from human induced pluripotent stem cells (iPSCs) is a powerful tool to identify tau-targeted therapeutics. However, such screens have been hampered by heterogeneous neuronal production, high cost and low yield, and multi-step differentiation procedures. We engineered an isogenic iPSC line that harbors an inducible neurogenin 2 transgene, a transcription factor that rapidly converts iPSCs to neurons, integrated at the AAVS1 locus. Using a simplified two-step protocol, we differentiated these iPSCs into cortical glutamatergic neurons with minimal well-to-well variability. We developed a robust high-content screening assay to identify tau-lowering compounds in LOPAC and identified adrenergic receptors agonists as a class of compounds that reduce endogenous human tau. These techniques enable the use of human neurons for high-throughput screening of drugs to treat neurodegenerative disease.
INTRODUCTION: Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative diseases that share clinical and neuropathological features. Furthermore, the most common genetic cause of both FTD and ALS is a GGGGCC (G4C2) repeat expansion in the C9orf72 gene. This repeat expansion leads to several abnormalities, including C9orf72 haploinsufficiency, the accumulation of repeat RNA, and the production of five aggregation-prone proteins composed of repeating dipeptides. However, the contribution of these abnormalities to disease pathogenesis remains unresolved. RATIONALE: Among the five dipeptide repeat proteins nonconventionally translated from expanded G4C2 repeats, proline-arginine (PR) repeat proteins [poly(PR) proteins] have proven especially toxic in various model systems. Their involvement in C9orf72-associated FTD and ALS (c9FTD/ALS) has nevertheless been questioned because poly(PR) pathology is relatively infrequent in c9FTD/ALS patient brains. Postmortem tissues, however, represent end-stage disease and do not necessarily reflect early events in the disease process. Therefore, we generated mice that express poly(PR) in the brain to evaluate the temporal consequences of its expression in a mammalian in vivo model. More specifically, we engineered mice to express green fluorescent protein (GFP)–conjugated (PR)50 (a 50-repeat PR protein) or GFP via intracerebroventricular administration of adeno-associated viral vectors and then performed behavioral, pathological, and transcriptomic characterizations of poly(PR) mice in comparison with control GFP mice. RESULTS: We found that ~60% of poly(PR)- expressing mice died by 4 weeks of age and had significantly decreased brain and body weights at death compared with age-matched GFP control mice. Poly(PR) mice that escaped premature death developedmotor andmemory impairments, likely as a consequence of their progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis. In investigating the mechanisms by which poly(PR) caused neurodegeneration and functional deficits, we found that poly(PR) localized to heterochromatin (highly condensed regions of transcriptionally silent chromatin) and caused abnormal histone H3 methylation, features that we also detected in brain tissues from patients with c9FTD/ALS. Additionally, we observed aberrations in nuclear lamins and heterochromatin protein 1α (HP1α), key proteins thatmaintain heterochromatin structure and regulate gene silencing. Nuclear lamina invaginations and decreased HP1a protein expression were seen in poly(PR)-positive cells in poly(PR) mice, and in vitro studies demonstrated that poly(PR) disrupted HP1α liquid phases. Because poly(PR)-induced histone H3 posttranslational modifications, lamin invaginations, and decreased HP1α levels could profoundly affect gene expression, we compared transcriptome profiles between control and poly(PR) mice. As well as analyzing differentially expressed genes, we examined repetitive element expression given that repetitive...
Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.
SignificanceLate-onset Alzheimer’s disease is the most common form of dementia. A rare hemizygous variant in a microglial-expressed gene, Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), significantly increases risk for late-onset Alzheimer’s disease. This variant is thought to cause loss of function, inducing TREM2 haploinsufficiency. The ramifications of TREM2 haploinsufficiency on microglial function and tau pathology are major gaps in the field. We find that, in contrast to the protective effects of complete TREM2 deficiency, TREM2 haploinsufficiency exacerbates tau pathology, inflammation, and atrophy at a late stage of disease in a mouse model of tauopathy. The differential effects of partial and complete loss of TREM2 are important considerations for TREM2-targeted therapeutic strategies.
CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSCderived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.2 4 systematic dissection of normal and disease states of neurons, and highlight the potential of interrogating human cell biology and gene function in iPSC-derived differentiated cell types. RESULTS Robust CRISPR interference in human iPSC-derived neuronsAs a first step towards a high-throughput screening platform in neurons, we developed a scalable CRISPRi-based strategy for robust knockdown of endogenous genes in homogeneous populations of human iPSC-derived neurons. We built on our previously described i 3 Neuron (i 3 N) platform, which enables large-scale production of iPSC-derived glutamatergic neurons. Central to this platform is an iPSC line with an inducible Neurogenin 2 (Ngn2) expression cassette in the AAVS1 safe-harbor locus (Fernandopulle et al., 2018;Wang et al., 2017). To enable stable CRISPRi in iPSC-derived neurons, we generated a plasmid (pC13N-dCas9-BFP-KRAB) to insert an expression cassette for CAG promoter-driven dCas9-BFP-KRAB into the CLYBL safe harbor locus, which enables robust transgene expression throughout neuronal differentiation at higher levels than the AAVS1 locus (Cerbini et al., 2015) ( Fig. 1A). We then integrated this cassette into our i 3 N iPSC line, and called the resulting monoclonal line CRISPRi-i 3 N iPSCs. A normal karyotype was confirmed for CRISPRi-i 3 N iPSCs ( Fig. S1A).To validate CRISPRi activity, we transduced these iPSCs with a lentiviral construct expressing an sgRNA targeting the transferrin receptor gene (TFRC). Knockdown of TFRC mRNA was robust in iPSCs and in i 3 Neurons for several weeks after differentiation (Fig. 1B,C). We also validated knockdown of three additional genes, UBQLN2 (Fig. 1D,E), GRN (Fig. 1F,G) and CDH2 (Fig. S1B) by qRT-PCR, Western blot, and/or immunofluorescence. Our platform thus enables potent CRISPRi knockdown of endogenous genes in iPSC-derived neurons.Since CRISPRn-associated DNA damage has been found to be highly toxic to iPSCs (Ihry ...
SummaryHuman astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations.
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